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The C-terminal domain of Escherichia coli Hfq is required for regulation.

Vecerek B, Rajkowitsch L, Sonnleitner E, Schroeder R, Bläsi U - Nucleic Acids Res. (2007)

Bottom Line: Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion.Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding.These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.

ABSTRACT
The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans-encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the gamma- and beta-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq(65)), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.

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Related in: MedlinePlus

Hfq65 is defective in auto- and riboregulation. (A) Relative translational efficiency of hfq131-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRhfq131; pACYC184), AM111F′ (pRhfq131; pAHfq) and AMF′111(pRhfq131; pAHfq65) grown in LB medium, respectively. (B) Relative translational efficiency of sodB-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRsodB; pACYC184), AM111F′ (pRsodB; pAHfq) and AM111F′ (pRsodB; pAHfq65) grown in M9 medium, respectively. The averaged β-galactosidase values normalized to mRNA levels obtained in the absence of Hfq was set to 1 (white bar). The values obtained in the presence of Hfqwt (black bar) and Hfq65 were normalized to the control. The experiment was performed in duplicate. The error bars represent standard deviations. Bottom: determination of the levels of Hfqwt and Hfq65 in the respective strains by quantitative immunoblotting (see Materials and Methods section). (C) Graphical representation of the σS levels in strain AM111F′ harbouring plasmid pUC18 (lane 1; control), pUHfq65 (lane 2; Hfq65) and pUH5 (lane 3; Hfqwt), respectively. The western blot analysis was carried with equal amounts of total cellular protein as described in Materials and Methods section. Only the relevant sections of the immunoblots (lower panels) showing the σS-and Hfq-specific bands are depicted. Quantification of the western blot was done with ImageQuant software. Values were normalized to the σS signal obtained in the presence of Hfqwt in strain AM111F′ (pUH5), which was set to 1. The results represent data from duplicate experiments. The error bars represent standard deviations.
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Figure 2: Hfq65 is defective in auto- and riboregulation. (A) Relative translational efficiency of hfq131-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRhfq131; pACYC184), AM111F′ (pRhfq131; pAHfq) and AMF′111(pRhfq131; pAHfq65) grown in LB medium, respectively. (B) Relative translational efficiency of sodB-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRsodB; pACYC184), AM111F′ (pRsodB; pAHfq) and AM111F′ (pRsodB; pAHfq65) grown in M9 medium, respectively. The averaged β-galactosidase values normalized to mRNA levels obtained in the absence of Hfq was set to 1 (white bar). The values obtained in the presence of Hfqwt (black bar) and Hfq65 were normalized to the control. The experiment was performed in duplicate. The error bars represent standard deviations. Bottom: determination of the levels of Hfqwt and Hfq65 in the respective strains by quantitative immunoblotting (see Materials and Methods section). (C) Graphical representation of the σS levels in strain AM111F′ harbouring plasmid pUC18 (lane 1; control), pUHfq65 (lane 2; Hfq65) and pUH5 (lane 3; Hfqwt), respectively. The western blot analysis was carried with equal amounts of total cellular protein as described in Materials and Methods section. Only the relevant sections of the immunoblots (lower panels) showing the σS-and Hfq-specific bands are depicted. Quantification of the western blot was done with ImageQuant software. Values were normalized to the σS signal obtained in the presence of Hfqwt in strain AM111F′ (pUH5), which was set to 1. The results represent data from duplicate experiments. The error bars represent standard deviations.

Mentions: Cultures of AM111F′ (pRhfq131) and AM111F′ (pRsodB-lacZ) co-transformed with the compatible plasmids pACYC184 (control), pAHfq and pAHfq65, respectively, were cultivated in LB or M9 medium (Figure 2) at 37°C. At an OD600 of 0.5, the plasmid-encoded genes were induced by addition of IPTG (1 mM). After 60 min, triplicate aliquots were taken for the β-galactosidase assays and for western blot analysis to verify Hfqwt or Hfq65 production. In parallel, samples were withdrawn for isolation of total RNA to determine the respective hfq-lacZ and sodB-lacZ mRNA levels. The β-galactosidase activity was determined from triplicate samples as described (45) and the total RNA was purified by the hot phenol method (48). The averaged β-galactosidase activities obtained with the pRhfq131 and pRsodB-lacZ constructs were normalized to the corresponding hfq-lacZ and sodB-lacZ mRNA levels (=relative translational efficiencies in Figure 2A and B). The corresponding lacZ mRNA levels were determined by primer extension with AMV reverse transcriptase (Promega GmbH, Germany) using 5 μg of total RNA primed with the lacZ-specific 5′-end labelled probe (5′-GGGAAGGGCGATCGGT-3′) and normalized to the 5S rRNA levels (internal control), which were determined using primer R25 (5′-GGTGGGACCACCGCGCTACGGCCGCCAGGC-3′). The signals were visualized by a PhosphoImager (Molecular Dynamics) and quantified by ImageQuant software. Two independent sets of experiments were performed.


The C-terminal domain of Escherichia coli Hfq is required for regulation.

Vecerek B, Rajkowitsch L, Sonnleitner E, Schroeder R, Bläsi U - Nucleic Acids Res. (2007)

Hfq65 is defective in auto- and riboregulation. (A) Relative translational efficiency of hfq131-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRhfq131; pACYC184), AM111F′ (pRhfq131; pAHfq) and AMF′111(pRhfq131; pAHfq65) grown in LB medium, respectively. (B) Relative translational efficiency of sodB-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRsodB; pACYC184), AM111F′ (pRsodB; pAHfq) and AM111F′ (pRsodB; pAHfq65) grown in M9 medium, respectively. The averaged β-galactosidase values normalized to mRNA levels obtained in the absence of Hfq was set to 1 (white bar). The values obtained in the presence of Hfqwt (black bar) and Hfq65 were normalized to the control. The experiment was performed in duplicate. The error bars represent standard deviations. Bottom: determination of the levels of Hfqwt and Hfq65 in the respective strains by quantitative immunoblotting (see Materials and Methods section). (C) Graphical representation of the σS levels in strain AM111F′ harbouring plasmid pUC18 (lane 1; control), pUHfq65 (lane 2; Hfq65) and pUH5 (lane 3; Hfqwt), respectively. The western blot analysis was carried with equal amounts of total cellular protein as described in Materials and Methods section. Only the relevant sections of the immunoblots (lower panels) showing the σS-and Hfq-specific bands are depicted. Quantification of the western blot was done with ImageQuant software. Values were normalized to the σS signal obtained in the presence of Hfqwt in strain AM111F′ (pUH5), which was set to 1. The results represent data from duplicate experiments. The error bars represent standard deviations.
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Figure 2: Hfq65 is defective in auto- and riboregulation. (A) Relative translational efficiency of hfq131-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRhfq131; pACYC184), AM111F′ (pRhfq131; pAHfq) and AMF′111(pRhfq131; pAHfq65) grown in LB medium, respectively. (B) Relative translational efficiency of sodB-lacZ mRNA in the absence of Hfq (white bar), in the presence of Hfqwt (black bar) and Hfq65 (grey bar) in strains AM111F′ (pRsodB; pACYC184), AM111F′ (pRsodB; pAHfq) and AM111F′ (pRsodB; pAHfq65) grown in M9 medium, respectively. The averaged β-galactosidase values normalized to mRNA levels obtained in the absence of Hfq was set to 1 (white bar). The values obtained in the presence of Hfqwt (black bar) and Hfq65 were normalized to the control. The experiment was performed in duplicate. The error bars represent standard deviations. Bottom: determination of the levels of Hfqwt and Hfq65 in the respective strains by quantitative immunoblotting (see Materials and Methods section). (C) Graphical representation of the σS levels in strain AM111F′ harbouring plasmid pUC18 (lane 1; control), pUHfq65 (lane 2; Hfq65) and pUH5 (lane 3; Hfqwt), respectively. The western blot analysis was carried with equal amounts of total cellular protein as described in Materials and Methods section. Only the relevant sections of the immunoblots (lower panels) showing the σS-and Hfq-specific bands are depicted. Quantification of the western blot was done with ImageQuant software. Values were normalized to the σS signal obtained in the presence of Hfqwt in strain AM111F′ (pUH5), which was set to 1. The results represent data from duplicate experiments. The error bars represent standard deviations.
Mentions: Cultures of AM111F′ (pRhfq131) and AM111F′ (pRsodB-lacZ) co-transformed with the compatible plasmids pACYC184 (control), pAHfq and pAHfq65, respectively, were cultivated in LB or M9 medium (Figure 2) at 37°C. At an OD600 of 0.5, the plasmid-encoded genes were induced by addition of IPTG (1 mM). After 60 min, triplicate aliquots were taken for the β-galactosidase assays and for western blot analysis to verify Hfqwt or Hfq65 production. In parallel, samples were withdrawn for isolation of total RNA to determine the respective hfq-lacZ and sodB-lacZ mRNA levels. The β-galactosidase activity was determined from triplicate samples as described (45) and the total RNA was purified by the hot phenol method (48). The averaged β-galactosidase activities obtained with the pRhfq131 and pRsodB-lacZ constructs were normalized to the corresponding hfq-lacZ and sodB-lacZ mRNA levels (=relative translational efficiencies in Figure 2A and B). The corresponding lacZ mRNA levels were determined by primer extension with AMV reverse transcriptase (Promega GmbH, Germany) using 5 μg of total RNA primed with the lacZ-specific 5′-end labelled probe (5′-GGGAAGGGCGATCGGT-3′) and normalized to the 5S rRNA levels (internal control), which were determined using primer R25 (5′-GGTGGGACCACCGCGCTACGGCCGCCAGGC-3′). The signals were visualized by a PhosphoImager (Molecular Dynamics) and quantified by ImageQuant software. Two independent sets of experiments were performed.

Bottom Line: Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion.Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding.These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.

ABSTRACT
The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans-encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the gamma- and beta-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq(65)), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.

Show MeSH
Related in: MedlinePlus